Craniofacial malformations can arise from genetic causes and/or environmental factors, including hypoxia in pregnancy. To understand the mechanisms underlying craniofacial malformations induced by hypoxia, I have designed a hypoxic chick embryo model by decreasing the level of oxygen in ovo. To test the correlation between hypoxia and craniofacial malformations, I will examine the role of developmental morphogenetic signaling and the cellular response to hypoxia via the hypoxia-inducible factor-1 (HIF-1) pathway and reactive oxygen species (ROS). Inspired by a recent clinical report of holoprosencephalic anomalies in an acardiac human twin fetus that was hypoxic, my preliminary studies demonstrate that hypoxia reduces survival and delays development in chick embryos, disrupts cell proliferation, and alters facial morphology. Hypoxia led to a roughly dose-dependent decrease in survival with increasing hypoxia, and the severity of developmental delay decreased with the age of survivors (ie, fewer survivors with more severe delay). Hypoxic embryos also showed a spectrum of cephalic and craniofacial malformations ranging from mild asymmetry and eye defects to more severe defects in frontonasal structures and exposed cephalic tissues, characteristic of holoprosencephaly. 2-dimensional geometric morphometrics showed significant abnormal facial shape variation in relation to centroid size and age, among individuals in hypoxic vs. normoxic control groups, and among hypoxic groups compared to the normoxic group. The morphometric data indicate that hypoxia leads to severe developmental delay, and to a lesser degree, malformations in embryos that are able to survive the hypoxic insult. Hypoxia disrupted cell proliferation. In early stages of development, apoptosis of neural crest progenitor cells was observed in varying degrees. How hypoxia causes craniofacial malformations is not understood. I hypothesize that hypoxia creates abnormal craniofacial morphology via Hif-11-mediated apoptosis and by decreased cell proliferation due to altered molecular signaling between the forebrain and frontonasal process. To test my hypothesis, I will determine the extent to which hypoxia generates significant morphological alterations by altering cell proliferation, cell death and molecular changes. In addition, I will undertake gain- and loss-of-function studies to define the relationship of hypoxia-adaptive molecular signaling to malformations. In my two Specific Aims, I will uncover the possible cellular and molecular mechanisms underlying malformations due to hypoxia, as well as the role of HIF-1 and ROS activity in cells under hypoxic stress. In Aim 1, I will determine the extent to which cellular and molecular changes due to hypoxia cause facial malformations that resemble holoprosencephaly, by quantifying changes in facial morphology and relating these changes to cellular behavior and molecular signaling;this will test directly the hypothesis that hypoxia produces holoprosencephaly. In Aim 2, I will test the relationship between HIF-11, ROS, and facial malformations, by assessing the extent to which HIF-11 is necessary and sufficient to cause defects resulting from hypoxia and by testing the hypothesis that the hypoxia sensor, ROS production by the electron transport chain in hypoxia, can be targeted to reduce mortality rates and reverse malformations. My findings will provide insights into how craniofacial malformations caused by hypoxia can be prevented and reversed with future therapeutic strategies. PUBLIC HEALTH RELEVANCE: How can an environmental stressor, such as oxygen deprivation, affect the development of craniofacial structures in the embryo? Answering this question is important for the prevention and treatment of craniofacial birth defects that at least partially arise from environmental stresses including the lack of oxygen during embryonic and fetal development, as well as for devising new therapies to reverse these malformations. This project aims to identify the cellular and molecular events that create craniofacial defects in a low-oxygen environment.